Abstract
Antipeptide antibody was raised in rabbit against the sequence (361-390) of RDC-8, the presumed adenosine A2A receptor cDNA from canine. The antibody titer was estimated by solid phase radioimmunoassay. Western blot analysis under reducing conditions identified a major 45 ± 1 kDa protein in bovine striatal membranes. This immunoreactive band was competed in the presence of excess peptide. Furthermore, the antibody recognized a single 45-kDa immunoreactive band in membranes from cells transfected with the recombinant human adenosine A2A receptors, whereas, fail to cross-react with membranes from cells transfected with recombinant rat A1 and human A3 receptors. Membranes from human and porcine coronary artery, ventricle, atria and platelets (human only) showed a major immunoreactive band at 45 ± 1 kDa size. Under nonreducing conditions, the migration patterns of the immunoreactive bands were not altered indicating the absence of interchain disulfide bond. The 45-kDa immunoreactive band co-migrated with 2-[4-(2-{2-[(4-aminophenyl)methylcarbonylamino]ethyl-aminocarbonyl}ethyl)phenyl]ethylamino-5′-Nethylcarboxamidoadenosine photoaffinity labeled A2A adenosine receptor using SANPAH as the photoaffinity cross-linker. We provide immunological evidence for the presence of A2A adenosine receptor in human cardiovascular tissues that exists as a 45-kDa monomeric protein. This study also presents evidence for the presence of A2Aadenosine receptor in ventricle and atria in both human and porcine.
Footnotes
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Send reprint requests to: Dr. S. Jamal Mustafa, Department of Pharmacology, School of Medicine, East Carolina University, Greenville, NC 27858-4354.
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↵1 This work was supported by National Heart, Lung and Blood Institute Grant HL-27339.
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↵2 Present address: Pfizer Central Research, Eastern Point Road, Groton, CT 06340.
- Abbreviations:
- PAPA-APEC
- 2-[4-(2-{2-[(4-aminophenyl)methylcarbonylamino]-ethyl-aminocarbonyl}ethyl)phenyl] ethylamino-5′-N-ethylcarboxamidoadenosine
- KLH
- keyhole limpet hemocyanin
- CGS-21680
- 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido adenosine
- SDS-PAGE
- sodium dodecyl sulfate polyacrylamide gel electrophoresis
- BSA
- bovine serum albumin
- PBS
- phosphate-buffered saline
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- Received November 17, 1997.
- Accepted April 13, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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